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Method for Polyunsaturated Fatty Acid Production Using Novel Cell Preservation Technique

a cell preservation and polyunsaturated fatty acid technology, applied in the field of microbial biomass production, can solve the problem of inability to achieve stable production outpu

Active Publication Date: 2008-02-14
NIPPON SUISAN KAISHA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] The present inventors conducted diligent research on the initial culturing stage conditions affecting the cultured cell growth phase during production of PUFA-containing fats and oils (triglycerides) and / or PUFA-containing phospholipids by culturing of microorganisms, and as a result discovered that by improving the transplanting conditions for hyphae or spores from previous steps, it is possible to increase the reproducibility of culturing and to achieve stable production of PUFA-containing fats and oils (triglycerides) and / or PUFA-containing phospholipids.
[0017] According to the invention, therefore, there is provided a method for production of PUFA-containing fats and oils (triglycerides) and / or PUFA-containing phospholipids and / or PUFA-containing cells is based on improved reproducibility of culturing and stable production of PUFA-containing fats and oils (triglycerides) and / or PUFA-containing phospholipids, the method being characterized by improving the transplanting conditions for hyphae or spores from previous steps.

Problems solved by technology

However, since these methods are affected by slight differences in the culturing conditions, it is not easy to ensure reproducibility of culturing and as a result, stable production output has not been possible to achieve.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Method for Cryopreservation of Spore Suspension

[0068]Mortierella alpina 1S-4 was used as an arachidonic acid-producing strain. Stationary culturing was carried out for 7 days at 25° C. at a slant in Czapek agar medium (adjusted to pH 6.0 and sterilized) provided in a test tube, and after confirming hyphal growth, the test tube was stored in a refrigerator (4° C.) for 10 days.

[0069] Sterilized water was added to the test tube and the mixture was well agitated to prepare a spore suspension. The spore suspension was appropriately diluted and coated onto a potato dextrose agar medium plate, and a colony counting method was used to count the spores in the spore suspension, giving a result of 1×106 spores / mL. Next, the spore suspension was diluted 100-fold with sterilized water. The diluted spore suspension, glycerin and water were mixed in the following proportion: diluted spore suspension:glycerin:water=1:1:8 (by volume) (the water and glycerin were premixed and sterilized). A 1 mL po...

example 2

Culturing Experiment, Difference in Reproducibility by Preservation Method

[0073] Culturing was carried out from a seed strain prepared by the method of Example 1 and Comparative Example 1 using M. alpina 1S-4.

[0074] After transferring 0.1 vol % of the preserved seed strain to medium at pH 6.3 containing 1.0% yeast extract and 2.0% glucose, seed culturing was initiated under conditions of 100 rpm reciprocal shaking, 28° C. temperature, and culturing was continued for 3 days.

[0075] Next, 25 L of medium at pH 6.3 containing 5.0% defatted soybean powder, 0.3% KH2PO4, 0.1% Na2SO4, 0.05% CaCl2.2H2O, 0.05% MgCl2.6H2O, 1.8% glucose and 0.1% soybean oil was prepared in a 50 L volume jar fermenter, and then 100 mL of the seed culture solution was transferred and culturing was initiated. under conditions of 92 rpm agitation, 26° C. temperature, 200 kPa internal pressure and 12.5 L / min airflow. During the culturing, glucose was added at the concentration shown in Table 1, and culturing was c...

example 3

Culturing Experiment, change in Productivity with Prolonged Preservation

[0078] Culturing was carried out from a seed strain prepared by the method of Example 1 and Comparative Example 1 using M. alpina 1S-4.

[0079] After transferring 0.1 vol % of the preserved seed strain to medium at pH 6.3 containing 1.0% yeast extract and 2.0% glucose, seed culturing was initiated under conditions of 100 rpm reciprocal shaking, 28° C. temperature, and culturing was continued for 3 days.

[0080] Next, 25 L of medium at pH 6.3 containing 1.0% yeast extract, 1.8% glucose and 0.1% soybean oil was prepared in a 50 L volume jar fermenter, and then 100 mL of the seed culture solution was transferred and culturing was initiated under conditions of 200 rpm agitation, 28° C. temperature, 150 kPa internal pressure and 25 L / min airflow. During the culturing, glucose was added at the concentration shown in Table 3, and culturing was continued for 7 days.

TABLE 3Time and concentration of glucose feeding (conc...

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PUM

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Abstract

A method for preservation of a microorganism capable of microbial production of a polyunsaturated fatty acid or a compound comprising a polyunsaturated fatty acid as a constituent fatty acid, which method comprises: (a) forming spores in a spore-forming medium at pH 4-7 containing a nutrient source comprising an inorganic salt and a saccharide; (b) suspending the spores obtained in (a) in sterilized water, or sterilized water containing a surfactant and / or an inorganic salt to prepare a spore suspension, and adding a cryoprotectant at 5-15% to prepare a cryopreserving spore suspension; and (c) preserving the cryopreserving spore suspension obtained in (b) at between −100° C. and −20° C.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for production of a microbial biomass which includes microorganisms that produce compounds comprising polyunsaturated fatty acids as constituent fatty acids, crude oils and / or crude phospholipids obtained by extraction from the biomass, and refined fats and oils and / or refined phospholipids obtained by refining of the crude oils and / or crude phospholipids, as well as to foods and beverages, therapeutic nutritional supplements, animal feeds and pharmaceuticals which incorporate the biomass and fats or oils (crude oils and / or refined oils) and / or phospholipids (crude phospholipids and / or refined phospholipids). BACKGROUND ART [0002] Biosynthesis of polyunsaturated fatty acids (hereinafter abbreviated as “PUFA”) in humans occurs for two representative series, the ω3 and ω6 series (where ω represents the number of the carbon atom having the first double bond, counting from the methyl group end of the fatty acid), and in th...

Claims

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Application Information

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IPC IPC(8): C12P7/64C12N1/20C12P7/6427C12P7/6431C12P7/6432C12P7/6434C12P7/6472C12P7/6481
CPCC12N1/04C12N1/14C12P7/6481C12P7/6472C12P7/6427C12P7/6434C12P7/6431C12P7/6432C12P7/64
Inventor HIGASHIYAMA, KENICHI
Owner NIPPON SUISAN KAISHA LTD
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